PROJECT SUMMARY Anemia is a common medical condition in preterm infants. Previous studies generated from this Program Project Grant (PPG) showed that the degree of anemia contributes significantly to the neurodevelopmental outcomes of these individuals- particularly with respect to deficits in learning and memory. Anemia and its therapies (red blood cell transfusion and Erythropoietin (Epo) administration) expose the rapidly developing brains of premature neonates to potential neuropathologies. More severe anemia secondary to restrictive transfusion protocols risks brain hypoxia and iron deficiency (ID). Conversely, liberal transfusion protocols place the brain at risk for toxicity from iron overload and suppression of endogenous Epo- a potential neuronal growth factor. Treatment with exogenous Epo may be neuroprotective, but may also shunt limited neonatal iron reserves into red blood cells, thereby restricting iron delivery to the brain and causing more severe brain ID. The hippocampus, a major brain region underlying recognition learning and memory, differentiates rapidly in the neonatal period. Proper structural differentiation requires adequate oxygen, iron and growth factors. Neonatal ID anemia has particularly profound effects on the developing hippocampus, affecting its genome, metabolome, structure, intracellular signaling pathways, electrophysiology and specific behavioral functions. These deficits manifest during the period of ID and remain into early adulthood in spite of iron repletion. Our outcome studies of premature infants with neonatal anemia have suggested that these infants have similar neurodevelopmental abnormalities. The goal of Project 4 is to provide experimental evidence in unique, developmentally synchronized mouse models to establish mechanisms that underlie brain injury due to neonatal anemia and its treatments. The overall research aim of Project 4 is to evaluate the effects of phlebotomy-induced anemia and Epo treatment with or without supplemental iron between postnatal days (P) P3 and 12 on learning and memory. We will do this by assessing the behavioral function, metabolome, structure and neuronal mammalian target of rapamycin (mTOR) signaling status of the mouse hippocampus during the period of anemia and again in young adulthood. mTOR is a highly conserved signaling cascade that senses changes in neuronal nutritional, oxygen and growth factor status and responds by adjusting protein translation and actin polymerization rates, which in turn determine neuronal structure and function. In Aim 1, we will use unique models generated by our laboratory to test whether phlebotomy-induced anemia and Epo treatment with low or high dose supplemental Fe impair recognition learning and memory behavior. In Aim 2, we will test whether these same experimental conditions alter the neurometabolome and structure of the hippocampus. In Aim 3 we will test hypotheses about how oxygen, iron and Epo regulate kinases and genes in the mTOR pathway since the fundamental mechanisms by which neurons are dependent on oxygen, iron and Epo for growth and development are not understood.